Most epithelial ovarian cancers are diagnosed postmenopausally, although the well-established epidemiological risk <em>factors</em> (parity, oral contraceptive use) are premenopausal. We hypothesized that accumulation of senescent <em>fibroblasts</em>, together with concomitant loss of presenescent <em>fibroblasts</em> within the ovarian cortex, promotes initiation and early development of ovarian cancer from ovarian surface epithelial (OSE) cells. To test this, we established immortalized OSE (IOSE) cell lines that mimic early neoplastic transformation by overexpressing the CMYC oncogene (IOSE(CMYC)) and normal ovarian presenescent (PSN) and senescent (SEN) <em>fibroblast</em> cell lines. We then evaluated the ability of PSN and SEN <em>fibroblasts</em> to transform IOSE and IOSE(CMYC) after coculture. SEN <em>fibroblasts</em> significantly enhanced neoplastic development of IOSE(CMYC) cells; there was an up to <em>15</em>-fold increase in migration of IOSE(CMYC) cells cocultured with SEN <em>fibroblasts</em> compared with PSN <em>fibroblasts</em>. Conditioned medium from SEN <em>fibroblasts</em> promoted anchorage-independent <em>growth</em> of IOSE(CMYC) cells. We studied <em>fibroblast</em>-epithelial cell interactions in heterotypic three-dimensional spheroid models. Dual immunohistochemical staining of spheroids for a proliferation marker (MIB-1) and cytokeratin-18 indicated that SEN <em>fibroblasts</em> induce approximately a five-fold increase in proliferation of IOSE(CMYC) cells relative to cocultures with PSN <em>fibroblasts</em>. SEN, but not PSN <em>fibroblasts</em>, also induced nuclear atypia in epithelial cells in three-dimensional spheroids. These data suggest for the first time that the accumulation of senescent, or loss of presenescent <em>fibroblasts</em>, can promote neoplastic development of partially transformed OSE cells in vitro and illustrates the power of using three-dimensional heterotypic modeling to gain better insights into the etiology underlying the development of epithelial ovarian cancer.

OBJECTIVE

Signaling through the fibroblast growth factor (FGF) pathway may account for tumor resistance to antiangiogenic therapies targeting the VEGF pathway. Here, dovitinib (TKI258), a potent oral inhibitor of FGF receptor, VEGF receptor (VEGFR), and platelet-derived growth factor receptor tyrosine kinases, is studied in a dose escalation trial.

METHODS

Patients with advanced or metastatic renal cell carcinoma (RCC) with predominant clear cell histology were treated with oral dovitinib 500 or 600 mg/day (5-days-on/2-days-off schedule).

RESULTS

Twenty heavily pretreated patients (median 3 prior regimens) were enrolled, with 16, 11, and 12 patients having previously received at least 1: VEGFR inhibitor, mTOR inhibitor, and immunotherapy, respectively. Fifteen and 5 patients were treated in 500- and 600-mg cohorts, respectively. Three patients experienced dose-limiting toxicities: grade 2 bradycardia (500 mg), grade 4 hypertensive crisis (600 mg), and grade 3 asthenia with grade 2 nausea and vomiting (600 mg). The most common adverse events related to dovitinib were nausea (75%), diarrhea (70%), vomiting (70%), and asthenia (50%), the majority of which were mild (grade 1 or 2), with grade 3 events 5% or less (except asthenia, 15%) and only one grade 4 event (hypertensive crisis). Two patients achieved a partial response (500 mg), and 12 patients had stable disease, including 2 patients with long lasting disease stabilizations (>1 year) in the 500-mg cohort.

CONCLUSIONS

Dovitinib was tolerable and showed antitumor activity at a maximum tolerated dose of 500 mg on a 5-days-on/2-days-off schedule in heavily pretreated RCC patients.

Farnesoid X receptor (FXR) is the master regulator of bile acid (BA) homeostasis because it controls BA synthesis, influx, efflux, and detoxification in the gut/liver axis. Deregulation of BA homeostasis has been linked to hepatocellular carcinoma (HCC), and spontaneous hepatocarcinogenesis has been observed in FXR-null mice. This dreaded liver neoplasm has been associated with both FXR gene deletion and BA-mediated metabolic abnormalities after inactivation of FXR transcriptional activity. In the present study, we addressed the hypothesis that intestinal selective FXR reactivation would be sufficient to restore the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>)/cholesterol-7alpha-hydroxylase (Cyp7a1) enterohepatic axis and eventually provide protection against HCC. To this end, we generated FXR-null mice with re-expression of constitutively active FXR in enterocytes (FXR(-/-)iVP16FXR) and corresponding control mice (FXR(-/-)iVP16). In FXR-null mice, intestinal selective FXR reactivation normalized BA enterohepatic circulation along with up-regulation of intestinal FXR transcriptome and reduction of hepatic BA synthesis. At 16 months of age, intestinal FXR reactivation protected FXR-null mice from spontaneous HCC development that occurred in otherwise FXR-null mice. Activation of intestinal FXR conferred hepatoprotection by restoring hepatic homeostasis, limiting cellular proliferation through reduced cyclinD1 expression, decreasing hepatic inflammation and fibrosis (decreased signal transducer and activator of transcription 3 activation and curtailed collagen deposition).

CONCLUSIONS

Intestinal FXR is sufficient to restore BA homeostasis through the FGF<em>15</em> axis and prevent progression of liver damage to HCC even in the absence of hepatic FXR. Intestinal-selective FXR modulators could stand as potential therapeutic intervention to prevent this devastating hepatic malignancy, even if carrying a somatic FXR mutation.

Bone contains various <em>growth</em> <em>factors</em>, including <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). The cellular origins of the <em>growth</em> <em>factors</em> found in bone are not known. We examined whether cultured fetal bovine bone cells synthesize FGF. These cells express characteristic markers of the osteoblast phenotype, including expression of bone Gla protein (osteocalcin) and mineralization. Heparin-Sepharose fractionation of cell extracts revealed that bone cells contained a basic FGF (bFGF)-like molecule, that displayed high affinity for heparin. The <em>growth</em> <em>factor</em> was mitogenic for adrenal cortex-derived endothelial cells and osteoblast-like bone cells. The major peak of biological activity corresponded to a peak of immunoreactive bFGF. When analyzed by Western blot, the active fractions contained a bFGF-like immunoreactive species with a mol wt of <em>15</em>,000, a mass identical to that of (des-1-<em>15</em>)bFGF. Based on RIA, the bone cell extract contained an estimated 95 ng bFGF/mg cell protein. An acidic FGF-like molecule with lower affinity for heparin was also present in the purified bone cell extracts, although at an approximately 10-fold lower concentration than bFGF. These results demonstrate that bone cells synthesize a mitogen indistinguishable from bFGF. In addition, Northern analysis revealed that the bone cells expressed 3.5- and 7.0-kilobase bFGF gene transcripts. We next examined whether the bone cell-derived bFGF is stored in a bioactive form in the extracellular matrix. Bone cells synthesized an extracellular matrix which was mitogenic for adrenal cortex-derived endothelial cells. However, if the bone cell extracellular matrix was preincubated with neutralizing anti-bFGF antibodies, its mitogenic properties were abolished. This suggests that bone cell-derived bFGF may function as an autocrine or paracrine mitogen via its deposition into the extracellular matrix of bone.

Brain tumour stem cells (BTSCs) are a small population of cells that has self-renewal, transplantation, multidrug resistance and recurrence properties, thus remain novel therapeutic target for brain tumour. Recent studies have shown that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists induce <em>growth</em> arrest and apoptosis in glioblastoma cells, but their effects on BTSCs are largely unknown. In this study, we generated gliospheres with more than 50% CD133+ BTSC by culturing U87MG and T98G human glioblastoma cells with epidermal <em>growth</em> <em>factor</em> (EGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In vitro treatment with PPARgamma agonist, <em>15</em>-Deoxy-Delta(12,14)-Prostaglandin J(2) (<em>15</em>d-PGJ2) or all-trans retinoic acid resulted in a reversible inhibition of gliosphere formation in culture. Peroxisome proliferator-activated receptor gamma agonists inhibited the proliferation and expansion of glioma and gliosphere cells in a dose-dependent manner. Peroxisome proliferator-activated receptor gamma agonists also induced cell cycle arrest and apoptosis in association with the inhibition of EGF/bFGF signalling through Tyk2-Stat3 pathway and expression of PPARgamma in gliosphere cells. These findings demonstrate that PPARgamma agonists regulate <em>growth</em> and expansion of BTSCs and extend their use to target BTSCs in the treatment of brain tumour.

OBJECTIVE

The diagnosis of mitochondrial disorders (MDs) is occasionally difficult because patients often present with solitary, or a combination of, symptoms caused by each organ insufficiency, which may be the result of respiratory chain enzyme deficiency. <em>Growth</em> differentiation <em>factor</em> <em>15</em> (GDF-<em>15</em>) has been reported to be elevated in serum of patients with MDs. In this study, we investigated whether GDF-<em>15</em> is a more useful biomarker for MDs than several conventional biomarkers.

METHODS

We measured the serum levels of GDF-<em>15</em> and fibroblast growth <em>factor</em> 21 (FGF-21), as well as other biomarkers, in 48 MD patients and in 146 healthy controls in Japan. GDF-<em>15</em> and FGF-21 concentrations were measured by enzyme-linked immunosorbant assay and compared with lactate, pyruvate, creatine kinase, and the lactate-to-pyruvate ratio. We calculated sensitivity and specificity and also evaluated the correlation based on two rating scales, including the Newcastle Mitochondrial Disease Rating Scale (NMDAS).

RESULTS

Mean GDF-<em>15</em> concentration was 6-fold higher in MD patients compared to healthy controls (2,711 ± 2,459 pg/ml vs 462.5 ± 141.0 pg/mL; p < 0.001). Using a receiver operating characteristic curve, the area under the curve was significantly higher for GDF-<em>15</em> than FGF-21 and other conventional biomarkers. Our date suggest that GDF-<em>15</em> is the most useful biomarker for MDs of the biomarkers examined, and it is associated with MD severity.

CONCLUSIONS

Our results suggest that measurement of GDF-<em>15</em> is the most useful first-line test to indicate the patients who have the mitochondrial respiratory chain deficiency.

Fish endure long periods of fasting and demonstrate an extensive capacity for rapid and complete recovery after refeeding. The underlying mechanisms through which nutrient intake activates an increase in somatic <em>growth</em> and especially in muscle <em>growth</em> is poorly understood. In this study we examined the expression profile of major muscle <em>growth</em> regulators in trout white muscle 4, 12, and 34 days after refeeding, using real-time quantitative RT-PCR. Mean insulin-like <em>growth</em> <em>factor</em> I (IGFI) mRNA level in muscle increased dramatically 8- and <em>15</em>-fold, 4 and 12 days, respectively, after refeeding compared to fasted trout. This declined thereafter. Conversely, only a weak but gradual increase in mean insulin-like <em>growth</em> <em>factor</em> II (IGFII) mRNA level was observed during refeeding. Inversely to IGFI, mean IGF receptor Ia (IGFRIa) mRNA level declined after ingestion of food. In contrast, IGF receptor Ib (IGFRIb) mRNA level was not affected by refeeding. Mean <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) mRNA level increased by 2.5-fold both 4 and 12 days after refeeding, whereas <em>fibroblast</em> <em>growth</em> <em>factor</em> 6 (FGF6) and myostatin mRNA levels were unchanged. Subsequent to IGFI and FGF2 gene activation, an increase in myogenin mRNA accumulation was observed at 12 days post-refeeding suggesting that an active differentiation of myogenic cells succeeds their proliferation. In conclusion, among the potential <em>growth</em> <em>factors</em> we examined in this study, IGFI and FGF2 were identified as candidate genes whose expression may contribute to muscle compensatory <em>growth</em> induced by refeeding.

Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish, and cafetière coffee, is the most potent cholesterol-elevating compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol, including cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many of which are regulated by the nuclear hormone receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR). Further studies demonstrate that cafestol is an agonist ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, sterol 12alpha-hydroxylase, and Na(+)-taurocholate cotransporting polypeptide in the liver of wild-type but not FXR null mice. Cafestol did not affect genes known to be up-regulated by FXR in the liver of wild-type mice, but did increase expression of the positive FXR-target genes intestinal bile acid-binding protein and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>) in the intestine. Because FGF<em>15</em> has recently been shown to function in an enterohepatic regulatory pathway to repress liver expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on liver gene expression. PXR-dependent gene regulation of cytochrome P450 3A11 and other targets by cafestol was also only seen in the intestine. Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF<em>15</em> gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR, and this may contribute to its impact on cholesterol homeostasis.

UNASSIGNED

Coronary artery calcification (CAC) is highly prevalent in dialysis-naive patients with chronic kidney disease (CKD). However, there are sparse data on the association of CAC with subsequent risk of cardiovascular disease and all-cause mortality in this population.

UNASSIGNED

To study the prospective association of CAC with risk of cardiovascular disease and all-cause mortality among dialysis-naive patients with CKD.

UNASSIGNED

The prospective Chronic Renal Insufficiency Cohort study recruited adults with an estimated glomerular filtration rate of 20 to 70 mL/min/1.73 m2 from 7 clinical centers in the United States. There were <em>15</em>41 participants without cardiovascular disease at baseline who had CAC scores.

UNASSIGNED

Coronary artery calcification was assessed using electron-beam or multidetector computed tomography.

UNASSIGNED

Incidence of cardiovascular disease (including myocardial infarction, heart failure, and stroke) and all-cause mortality were reported every 6 months and confirmed by medical record adjudication.

UNASSIGNED

During an average follow-up of 5.9 years in <em>15</em>41 participants aged 21 to 74 years, there were 188 cardiovascular disease events (60 cases of myocardial infarction, 120 heart failures, and 27 strokes; patients may have had >1 event) and 137 all-cause deaths. In Cox proportional hazards models adjusted for age, sex, race, clinical site, education level, physical activity, total cholesterol level, high-density lipoprotein cholesterol level, systolic blood pressure, use of antihypertensive treatment, current cigarette smoking, diabetes status, body mass index, C-reactive protein level, hemoglobin A1c level, phosphorus level, troponin T level, log N-terminal pro-B-type natriuretic peptide level, <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 level, estimated glomerular filtration rate, and proteinuria, the hazard ratios associated with per 1 SD log of CAC were 1.40 (95% CI, 1.16-1.69; P < .001) for cardiovascular disease, 1.44 (95% CI, 1.02-2.02; P = .04) for myocardial infarction, 1.39 (95% CI, 1.10-1.76; P = .006) for heart failure, and 1.19 (95% CI, 0.94-1.51; P = .<em>15</em>) for all-cause mortality. In addition, inclusion of CAC score led to an increase in the C statistic of 0.02 (95% CI, 0-0.09; P < .001) for predicting cardiovascular disease over use of all the above-mentioned established and novel cardiovascular disease risk <em>factors</em>.

UNASSIGNED

Coronary artery calcification is independently and significantly related to the risks of cardiovascular disease, myocardial infarction, and heart failure in patients with CKD. In addition, CAC improves risk prediction for cardiovascular disease, myocardial infarction, and heart failure over use of established and novel cardiovascular disease risk factors among patients with CKD; however, the changes in the C statistic are small.

The current studies show FGF<em>15</em> signaling decreases hepatic forkhead transcription <em>factor</em> 1 (FoxO1) activity through phosphatidylinositol (PI) 3-kinase-dependent phosphorylation. The bile acid receptor FXR (farnesoid X receptor) activates expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>15</em> in the intestine, which acts through hepatic FGFR4 to suppress cholesterol-7alpha hydroxylase (CYP7A1) and limit bile acid production. Because FoxO1 activity and CYP7A1 gene expression are both increased by fasting, we hypothesized CYP7A1 might be a FoxO1 target gene. Consistent with recently reported results, we show CYP7A1 is a direct target of FoxO1. Additionally, we show that the PI 3-kinase pathway is key for both the induction of CYP7A1 by fasting and the suppression by FGF<em>15</em>. FGFR4 is the major hepatic FGF receptor isoform and is responsible for the hepatic effects of FGF<em>15</em>. We also show that expression of FGFR4 in liver was decreased by fasting, increased by insulin, and reduced by streptozotocin-induced diabetes, implicating FGFR4 as a primary target of insulin regulation. Because insulin and FGF both target the PI 3-kinase pathway, these observations suggest FoxO1 is a key node in the convergence of FGF and insulin signaling pathways and functions as a key integrator for the regulation of glucose and bile acid metabolism.

The recruitment of primordial follicles to initiate folliculogenesis determines the population of developing follicles available for ovulation and directly regulates female reproductive efficiency. In the current study, a floating organ culture system was used to examine the progression of primordial (stage 0) follicles to developing (stages 1-4) follicles in 4-day-old pre-pubertal rat ovaries. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was found to induce primordial follicle development similar to what has been demonstrated for kit ligand/stem cell <em>factor</em> (KL). The bFGF-treated ovaries contained 85% developing follicles compared with 50% developing follicles for control untreated organ cultures. Correspondingly, the number of primordial follicles in bFGF-treated ovaries decreased to <em>15</em>% of the total compared with 45% for controls. A bFGF neutralizing antibody was found to decrease the small amount of spontaneous follicle development that occurs during the organ culture. Basic FGF was localized to primordial and early developing follicles by immunocytochemistry and was primarily observed in the oocytes. Treatment of bovine ovarian theca cells and stroma cells with bFGF was found to promote cell <em>growth</em>. Basic FGF produced by the oocyte in early stage follicles appears to act on adjacent somatic cells to promote cell <em>growth</em> and development. Basic FGF, like KL, appears to be a primordial follicle-inducing <em>factor</em>. In summary, bFGF can regulate primordial follicle development that directly influences female reproductive efficiency.

OBJECTIVE

To study lens epithelial cell (LEC) regulation after experimental cataract surgery in rabbits. The effect of aqueous humor (AqH) on LECs in vitro was investigated and the AqH concentrations of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) were determined.

METHODS

Aqueous humor was aspirated immediately before, and 1, 5, 10, 15, and 30 days after cataract surgery (n=8 each day). The effect of AqH on LEC proliferation was investigated by the incorporation of [3H]thymidine. The following concentrations were determined in AqH: bFGF, active and total TGF-beta, leukocytes, and total protein.

RESULTS

Aqueous humor collected after surgery triggered LEC growth on each occasion, except on (postoperative) day 30, in contrast to the effect of normal AqH. Lens epithelial cell proliferation peaked on day 1, displaying a sixfold increase. The bFGF concentration in AqH increased 6 to 10-fold after surgery and remained high throughout the experimental period. The AqH concentration of active TGF-beta decreased sevenfold 1 day after surgery. It then returned to normal levels on day 15. Total TGF-beta in postoperative AqH was twice as high as that in normal AqH on all days. Lens epithelial cell proliferation correlated with protein (r=0.685), leukocytes (r=0.565), and active TGF-beta (r=-0.418).

CONCLUSIONS

The stimulating effect of postoperative AqH on LEC proliferation may be caused by a reduction in the concentration of TGF-beta.

<em>Growth</em> curves for multicellular spheroids of <em>15</em> different tumor and normal cell lines were characterized by doubling times which decreased with increasing <em>growth</em> until a stable saturation was attained. In spite of the identical and constant conditions during <em>growth</em>, the size at saturation varied by <em>factors</em> of 67 in spheroid volume and 75 in cell content. These saturation sizes showed no correlation with the monolayer doubling times or clonogenic efficiencies, the initial spheroid <em>growth</em> rate or clonogenic capacity at saturation, the cell packing density, or the species of origin and type of cell line. There was a strong correlation between the maximal spheroid size and the size at which necrosis initially developed, suggesting control by necrosis. Crude extracts prepared from spheroids with extensive necrosis showed dose-dependent cytostatic and cytotoxic activities against monolayer cultures, while similar extracts from spheroids without necrosis had little effect. This activity was also detected in the culture medium to which the large spheroids had been exposed prior to preparation of extracts, suggesting that the responsible <em>factor</em>(s) can diffuse through the spheroid. The extract from spheroids of one cell line inhibited the <em>growth</em> and clonogenicity of four other cell lines, including human diploid <em>fibroblasts</em>. DNA content profiles measured during exposure to this extract showed that the cytostatic effect was not due to the arrest of cells in a specific cell cycle phase. The cell volumes were increased during culture in medium containing the extract from spheroids with extensive necrosis. These data support the hypothesis that <em>growth</em> saturation in spheroids is regulated by <em>factors</em> produced, released, or activated during the process of necrosis and suggest that these toxic <em>factors</em> have potential therapeutic use.

Previously we proposed that Schwann cell development from the neural crest is a two-step process that involves the generation of one main intermediate cell type, the Schwann cell precursor. Until now Schwann cell precursors have only been identified in the rat, and much remains to be learned about these cells and how they generate Schwann cells. Here we identify this cell in the mouse and analyze its transition to form Schwann cells in terms of timing, molecular expression, and extracellular signals and intracellular pathways involved in survival, proliferation, and differentiation. In the mouse, the transition from precursors to Schwann cells takes place 2 days earlier than in the rat, i.e., between embryo days 12/13 and <em>15</em>/16, and is accompanied by the appearance of the 04 antigen and the establishment of an autocrine survival circuit. Beta neuregulins block precursor apoptosis and support Schwann cell generation in vitro, a process that is accelerated by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> 2. The development of Schwann cells from precursors also involves a change in the intracellular survival signals utilized by neuregulins: To block precursor death neuregulins need to signal through both the mitogen-activated protein kinase and the phosphoinositide-3-kinase pathways although neuregulins support Schwann cell survival by signaling through the phosphoinositide-3-kinase pathway alone. Last, we describe the generation of precursor cultures from single 12-day-old embryos, a prerequisite for culture studies of genetically altered precursors when embryos are non-identical with respect to the transgene in question.

OBJECTIVE

To characterize the effects of small applied electric fields (EFs) (100 to 250 mV/ mm) on cultured bovine corneal epithelial cell (CEC) sheets and to determine how EFs interact with other environmental cues in directing CEC sheet migration.

METHODS

Primary cultures of bovine CECs were exposed to EFs in medium with or without serum, epithelial growth factor, basic fibroblast growth factor, or transforming growth factor-beta 1. Cell sheet migration was traced using an image analyzer.

RESULTS

Cell sheets migrated toward the cathode (negative pole). The directional migration was voltage dependent, and, at low field strength (up to 200 mV/mm), it required serum in the medium. Sheets showed no migration responses up to 200 mV/mm in serum-free medium, whereas those in medium with serum showed evident migration toward the cathode, at an average rate of approximately 15 microns/h (n = 15 approximately 20) at 150 mV/mm. When serum was present, the threshold was below 100 mV/mm, very close to the measured wound field strength (approximately 42 mV/mm). After supplementing serum-free medium with individual growth factors or with combinations of epithelial growth factor, basic fibroblast growth factor, and transforming growth factor-beta 1, significant restoration of cathode-directed migration occurred at 150 mV/ mm. Lamellipodia were abundant at the leading edges of migrating sheets, extending the area of sheets covered. The extension of cell membranes toward the cathode was more prominent in cell sheets than in single cells.

CONCLUSIONS

The endogenous EFs generated by wounded cornea could play an important role by interacting with other environmental factors to promote changes in shape and in directed migration of CEC sheets.

Matrix accumulation in the renal tubulointerstitium is predictive of a progressive decline in renal function. Transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta(1)) and, more recently, connective tissue <em>growth</em> <em>factor</em> (CTGF) are recognized to play key roles in mediating the fibrogenic response, independently of the primary renal insult. Further definition of the independent and interrelated effects of CTGF and TGF-beta(1) is critical for the development of effective antifibrotic strategies. CTGF (20 ng/ml) induced fibronectin and collagen IV secretion in primary cultures of human proximal tubule cells (PTC) and cortical <em>fibroblasts</em> (CF) compared with control values (P < 0.005 in all cases). This effect was inhibited by neutralizing antibodies to either TGF-beta or to the TGF-beta type II receptor (TbetaRII). TGF-beta(1) induced a greater increase in fibronectin and collagen IV secretion in both PTC (P < 0.01) and CF (P < 0.01) compared with that observed with CTGF alone. The combination of TGF-beta(1) and CTGF was additive in their effects on both PTC and CF fibronectin and collagen IV secretion. TGF-beta(1) (2 ng/ml) stimulated CTGF mRNA expression within 30 min, which was sustained for up to 24 h, with a consequent increase in CTGF protein (P < 0.05), whereas CTGF had no effect on TGF-beta(1) mRNA or protein expression. TGF-beta(1) (2 ng/ml) induced phosphorylated (p)Smad-2 within <em>15</em> min, which was sustained for up to 24 h. CTGF had a delayed effect on increasing pSmad-2 expression, which was evident at 24 h. In conclusion, this study has demonstrated the key dependence of the fibrogenic actions of CTGF on TGF-beta. It has further uniquely demonstrated that CTGF requires TGF-beta, signaling through the TbetaRII in both PTCs and CFs, to exert its fibrogenic response in this in vitro model.

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three <em>factors</em>: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF, 100 ng/ml) or epidermal <em>growth</em> <em>factor</em> (EGF, 50 ng/ml). The omission of any of these three <em>factors</em> from the synthetic medium results in a lower <em>growth</em> rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell <em>growth</em>. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent <em>15</em> generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell <em>growth</em> but also in increasing the longevity of the cultures.

Egr-1 is an early <em>growth</em> response gene that encodes a protein with three zinc fingers and is involved in transcriptional regulation. In adult heart myocytes, in contrast to c-fos and c-myc, high levels of Egr-1 mRNA expression have been shown. Here we report that Egr-1 transactivates rat cardiac alpha-MHC gene expression. In serum-starved primary cultures of 18-day-old fetal rat heart myocytes, addition of serum evoked expression of both Egr-1 and alpha-MHC gene transcripts. Inclusion of 10 microM cycloheximide in these cultures for 48 h caused a greater increase in Egr-1 mRNA, whereas the expression of alpha-MHC transcripts was ablated. To examine the involvement of Egr-1 in alpha-MHC induction, we transfected primary cultures of cardiac myocytes with plasmids pCMVEgr-1 (Egr-1 expression vector) and pMP3.3CAT containing -2.9- to +0.42-kilobase sequences of the alpha-MHC gene fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of pCMVEgr-1 stimulated expression of pMP3.3CAT 10-<em>15</em>-fold. Furthermore, pCMVEgr-1 also stimulated expression of the endogenous alpha-MHC gene in primary cultures of cardiac myocytes. Transactivation of pMP3.3CAT expression by pCMVEgr-1 was also observed by transfecting the myogenic cell line Sol 8, but not in L6E9 cells or in NIH3T3 <em>fibroblasts</em>. By creating progressive 5' deletions of the alpha-MHC gene, we found that the region extending between -1698 and -1283 base pairs is necessary for Egr-1-induced expression of the alpha-MHC/CAT construct. These results define a physiological target for the Egr-1 transcription <em>factor</em> and delineate a novel mechanism for regulation of the alpha-MHC gene.

Recent studies have implicated the hydrolysis of phosphoinositides and phosphatidylcholine in agonist-stimulated events. The potent mitogen, alpha-thrombin, stimulates the generation of diglycerides in a biphasic and sustained manner in IIC9 <em>fibroblasts</em> (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using measurements of radiolabeled headgroup release and molecular species analysis, we previously determined that alpha-thrombin generates diglycerides through the hydrolysis of both the phosphoinositides and phosphatidylcholine at early times (<em>15</em> s), and at later times (greater than or equal to 5 min) through the hydrolysis of primarily, if not exclusively, phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738). In contrast, IIC9 <em>fibroblasts</em> respond to the mitogenic treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b) epidermal <em>growth</em> <em>factor</em> by increasing their levels of diglycerides in a monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we have analyzed the molecular species of the diglycerides generated by these two different treatments and have also examined the lipid response of IIC9 <em>fibroblasts</em> to platelet-derived <em>growth</em> <em>factor</em>. Based on both the molecular species analyses and the release of radiolabeled head-groups, all three of these different mitogenic treatments generate diglycerides primarily through the stimulation of phosphatidylcholine hydrolysis. However, while similar, the molecular species profiles of the diglycerides generated by these three treatments are not identical to the molecular species profile of total cellular phosphatidylcholine. In addition, the molecular species profiles of the diglycerides generated by these three mitogenic treatments greatly resemble each other, with significant differences between any two profiles occurring in at most one molecular species. This finding differs from that seen with alpha-thrombin stimulation alone, where the molecular species profile of the diglycerides generated following 5 min of alpha-thrombin stimulation is nearly identical to the molecular species profile of total cellular phosphatidylcholine. These data support the possibility of hormone-sensitive phosphatidylcholine pools or selective diglyceride metabolism.

Interaction of the nerve <em>growth</em> <em>factor</em> (NGF) receptor/Trk with cellular substrates was investigated by transient co-overexpression in human 293 <em>fibroblasts</em> using ET-R, a chimeric receptor consisting of the epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) extracellular ligand binding domain and the Trk transmembrane and intracellular signal-generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C gamma (PLC gamma), ras GTPase-activating protein (GAP) and the non-catalytic subunit of phosphatidylinositol-3'-kinase, p85, in a ligand-dependent manner. Deletion of <em>15</em> C-terminal amino acids, including tyrosine 785 (Y-785) abrogated receptor and substrate phosphorylation activities. Mutation of Y-785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET-YF phosphorylation of PLC gamma was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine-phosphorylated synthetic C-terminal peptide competitively inhibited Trk cytoplasmic domain association with PLC gamma. Thus, the short C-terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y-785 is a major and selective interaction site for PLC gamma.

OBJECTIVE

Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1 alpha expression in different atherosclerotic plaque phenotypes.

RESULTS

HIF-1 alpha expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1 alpha was associated with the presence of a large extracellular lipid core (P=0.03) and macrophages (P=0.02). HIF-1 alpha co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1 alpha. In addition, a strong association was observed between expression levels of HIF-1 alpha and VEGF (P=0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1 alpha staining than in plaques with moderate or heavy HIF-1 alpha staining (P=0.03). In human macrophages, lipopolysaccharide activation induced HIF-1 alpha expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1 alpha mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation.

CONCLUSIONS

In atherosclerotic plaque, the transcription factor HIF-1 alpha is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1 alpha expression is upregulated in activated macrophages under normoxic conditions.

BACKGROUND

Activation of Bax and Bak, which act to permeabilize the mitochondrial membrane, is an essential step in the cell death response and therefore in the suppression of tumorigenesis. However, the mechanisms that regulate activation are poorly understood.

METHODS

Bax and Bak activation (conformational change and dimerization) was monitored in Rat-1 fibroblasts and human cancer cells subjected to endoplasmic reticulum (ER) stress, DNA damage, or tumor necrosis factor-alpha (TNF-alpha) treatment. Pharmacologic inhibitors of reactive oxygen species production, electron transport in the respiratory chain, oxidative phosphorylation, and appropriate controls were used to identify potential modes by which Bax and Bak activation and the cell death response are controlled. The oligomerization state of Bax and Bak was determined by cross-linking and subsequent immunoblot analysis; Bax conformational change was analyzed by immunoprecipitation and immunoblotting with an antibody specific for the active conformation. Cell death was evaluated by dye exclusion.

RESULTS

In both fibroblasts and human cancer cells subjected to cell death stimuli, inhibition of oxidative phosphorylation by use of antimycin A or oligomycin prevented ER stress-, DNA damage-, and TNF-alpha-induced Bax and Bak activation and cell death (UV-induced Rat-1 cell death at 15 hours: control, mean = 33.6%, 95% confidence interval [CI] = 18.8% to 48.4%; antimycin A, mean = 10.0%, 95% CI = 0% to 21.7%; oligomycin, mean = 13.1%, 95% CI = 5.7% to 20.5%; tunicamycin-induced MCF-7 cell death at 9 hours: control, mean = 29.2%, 95% CI = 21.6% to 36.8%; antimycin A, mean = 15.3%, 95% CI = 0.8% to 29.8%; oligomycin, mean = 11.5%, 95% CI = 3.9% to 19.1%; TNF-alpha-induced MCF-7 cell death at 6 hours: control, mean = 24.0%, 95% CI = 12.6% to 35.4%; antimycin A, mean = 8.9%, 95% CI = 3.9% to 13.9%; oligomycin, mean = 13.3%, 95% CI = 10.4% to 16.2%). Increasing and decreasing glycolytic adenosine triphosphate production, by adding glucose and 2-deoxy-D-glucose to the cell growth medium, respectively, neither reversed nor recapitulated, respectively, the effect of compromised oxidative phosphorylation on Bax and Bak activation.

CONCLUSIONS

Oxidative phosphorylation is required for the activation of Bax and Bak and cell death triggered by disparate death stimuli. The reliance of tumor cells on glycolysis in preference to oxidative phosphorylation even under normoxic conditions (Warburg effect) may therefore be a potential means by which these cells evade programmed cell death.

OBJECTIVE

Bile acid (BA) malabsorption of moderate severity is reported in 32% of patients with chronic unexplained diarrhea, including diarrhea-predominant irritable bowel syndrome (IBS-D). We hypothesized that variants of genes regulating hepatic BA synthesis play a role in IBS-D.

METHODS

In 435 IBS and 279 healthy subjects, we tested individual associations of <em>15</em> common single nucleotide polymorphisms (SNPs) from 7 genes critical to BA homeostasis with symptom-based subgroups using dominant genetic models. In a subset of 238 participants, we tested association with colonic transit. SNP-SNP interactions were investigated based on known protein interactions in BA homeostasis. The function of SNP rs17618244 in Klothoβ (KLB) was evaluated using a protein stability assay in HEK293 cells.

RESULTS

SNP rs17618244 (Arg728Gln in KLB) is associated with colonic transit at 24 hours. G allele (Arg728) compared with A allele (Gln728) is associated with accelerated colonic transit (P=.0007) in the overall cohort; this association was restricted to IBS-D (P=.0018). Interaction tests of KLB rs17618244 with 3 nonsynonymous SNPs of fibroblast growth factor receptor 4 (FGFR4) revealed that rs1966265 (Val10Ile) and rs351855 (Gly388Arg) modulate rs1768244's association with colonic transit in IBS-D (P=.0025 and P=.0023, respectively). KLB Arg728 significantly reduced protein stability compared with KLB Gln728, demonstrating KLB rs17618244's functional significance. No significant associations with symptom-based subgroups of IBS were detected.

CONCLUSIONS

A functional KLB gene variant mediating protein stability associates with colonic transit in IBS-D. This association is modulated by 2 genetic variants in FGFR4. The FGF19-FGFR4-KLB pathway links regulation of BA synthesis to colonic transit in IBS-D.

BACKGROUND

Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling.

RESULTS

Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO2 = 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the <em>growth</em> mediators PDGF-B, TGF-beta, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, <em>15</em> genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription <em>factors</em>. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media.

CONCLUSIONS

Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.